In 1981, acquired immune deficiency syndrome (AIDS) was identified as a disease that severely compromises the human immune system. In 1983, the etiological cause of AIDS was determined to be the human immunodeficiency virus (HIV). Reverse transcription is a common feature of retrovirus replication. Viral replication requires a virally encoded reverse transcriptase to generate DNA copies of viral sequences by reverse transcription of the viral RNA genome. Reverse transcriptase, therefore, is a clinically relevant target for the chemotherapy of retroviral infections because the inhibition of virally encoded reverse transcriptase would interrupt viral replication.
A number of compounds are effective in the treatment of the human immunodeficiency virus (HIV) which is the retrovirus that causes progressive destruction of the human immune system with the resultant onset of AIDS. Effective treatment through inhibition of HIV reverse transcriptase is known for both nucleoside based inhibitors and non-nucleoside based inhibitors. Nucleoside based HIV inhibitors in the treatment of AIDS. In 1985, it was reported that the synthetic nucleoside 3′-azido-3′-deoxythymidine (AZT) inhibits the replication of human immunodeficiency virus. Since then, a number of other synthetic nucleosides, including 3′-azidoguanosine (AZG), 2′,3′-dideoxycytidine (ddC), 2′,3′-dideoxyadenosine (ddA), 3′-fluoro-3′-deoxythymidine (FDDT) and 2′,3′-dideoxyinosine (ddI) have been proven to be effective against HIV. After cellular phosphorylation to the 5′-triphosphate by cellular kinases, these synthetic nucleosides are incorporated into a growing strand of viral DNA, causing chain termination due to the absence of the 3′-hydroxyl group. They can also inhibit the viral enzyme reverse transcriptase.
The success of various synthetic nucleosides in inhibiting the replication of HIV in vivo or in vitro has led a number of researchers to design and test nucleosides that substitute a heteroatom for the carbon atom at the 3′-position of the nucleoside. Norbeck, et al., disclosed that (±)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine (referred to as (±)-dioxolane-T) exhibits a modest activity against HIV (EC50 of 20 μM in ATH8 cells), and is not toxic to uninfected control cells at a concentration of 200 μM. Tetrahedron Letters 30 (46), 6246, (1989). European Patent Application Publication No. 0 337 713 and U.S. Pat. No. 5,041,449, assigned to BioChem Pharma, Inc., disclose racemic 2-substituted-4-substituted-1,3-dioxolanes that exhibit antiviral activity.
U.S. Pat. No. 5,047,407 and European Patent Application Publication No. 0 382 526, also assigned to BioChem Pharma, Inc., disclose that a number of racemic 2-substituted-5-substituted-1,3-oxathiolane nucleosides have antiviral activity, and specifically report that the racemic mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as BCH-189) has approximately the same activity against HIV as AZT, and little toxicity. BCH-189 has also been found to inhibit the replication of AZT-resistant HIV isolates in vitro from patients who have been treated with AZT for longer than 36 weeks. The (−)-enantiomer of the β-isomer of BCH-189, known as 3TC, which is highly potent against HIV and exhibits little toxicity, has been approved for the treatment of HIV in humans by the U.S. Food and Drug Administration in combination with AZT.
It has also been disclosed that cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”) has potent HIV activity. Schinazi, et al., “Selective Inhibition of Human Immunodeficiency viruses by Racemates and Enantiomers of cis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosine” Antimicrobial Agents and Chemo-therapy, November 1992, page 2423-2431. See also U.S. Pat. Nos. 5,210,085; 5,204,466, WO 91/11186, and WO 92/14743.
A number of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides have been shown to have potent anti-HIV-1 activity. 2′,3′-Dideoxy-2′,3′-didehydro-thymidine (“D4T”; also referred to as 1-(2,3-dideoxy-β-D-glycero-pent-2-eno-furanosyl)thymine) is currently sold for the treatment of HIV under the name Stavudine by Bristol Myers Squibb. Other D4 nucleosides that have been tested include 2′,3′-dideoxy-2′,3′-didehydro-cytidine (“D4C”), 2′,3′-dideoxy-2′,3′-didehydro-uridine (“D4U”), 2′,3′-dideoxy-2′,3′-didehydro-adenosine (“D4A”), 2′,3′-dideoxy-2′,3′-didehydro-inosine (“D4I”), and 2′,3′-dideoxy-2′,3′-didehydro-guanosine (“D4G”).
Other 2′,3′-dideoxy-2′,3′-didehydronucleosides have been reported to be effective against HIV and/or HBV. Starrett, Jr. et al., in U.S. Pat. Nos. 4,904,770, and 5,130,421, and Skonezny et al., in U.S. Pat. No. 5,539,099, disclose processes for the preparation of compounds of formula: wherein X and Z are selected from N or CH;                Y is selected from CR2 or N        R2 is selected from H, unsubstituted and halo-substituted alkyl having formula CnH2nA and alkenyl having the formula (CH2)m—CH═CHA wherein m=0, 1, 2 or 3 and n is 1, 2 or 3; and A is H, F, Cl, Br and I; and        R4 is NH2 or OH, useful as antiviral agents against HIV.        
Belica et al., in U.S. Pat. No. 4,900,828, disclose a process for the preparation of a compound of formula wherein R is a substituted or unsubstituted 2-acetoxy-2-methyl-propanoyl, 2-acetoxypropanoyl or 2-acetoxybenzoyl, optionally substituted with a lower alkyl, aryl or aralkyl;                R1 is a substituted or unsubstituted lower alkyl, aryl, aralkyl optionally substituted with halogen, alkyl, nitro or alkoxy;        which is used to prepare 2′,3′-dideoxynucleosides such as 2,3′-dideoxycytidine (ddC). See also Manchand P. S., J. Org. Chem., 1992, 57, 3473.        
In particular, β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine or β-D-D4FC, having the structure exhibits potent anti-HIV activity in vitro and demonstrates no cross-resistance with all approved anti-HIV agents. See Schinazi et al., J. Med. Chem. 1999, 42,859-867.
Also see Schinazi et al., in U.S. Pat. No. 5,703,058 which discloses a method and composition for compounds of formula wherein Y is O, S, CH2, CHF, CF2;                Z is O, S or Se;        R1 is H or F;        R2 is H, OH, C1-C6 alkyl or C(O)C1-C6alkyl; and        R3 is H, C(O)C1-C6alkyl, alkyl or mono-, di- or tri-phosphate;        for the treatment of HIV and HBV infections in humans and other host animals.        
U.S. Pat. No. 6,232,300 and International Patent Application No. PCT/US96/00965, published as WO 96/22778 discloses a method for the treatment of HIV using β-D-D4FC. U.S. Pat. No. 5,703,058 discloses a method for the treatment of HIV and HBV infection that includes administering an effective amount of β-L-D4FC in combination or alternation with cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane, 9-[4-(hydroxy-methyl)-2-cyclopenten-1-yl)-guanine (carbovir), 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon, 3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI), 2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine (L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T). U.S. Pat. No. 5,905,070 discloses a method for the treatment of HIV and HBV infection that includes administering an effective amount of β-D-D4FC in combination or alternation with cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, cis-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane, 9-[4-(hydroxymethyl)-2-cyclopenten-1-yl)-guanine (carbovir), 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), interferon, 3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI), 2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine (L-FMAU) or 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T).
European Patent Application Publication No. 0 409 227 A2 filed by Ajinomoto Co., Inc., discloses β-D-D4FC (Example 2) and its use to treat hepatitis B. Netherlands Patent No. 8901258 filed by Stichting Rega V. Z. W. discloses generally 5-halogeno-2′,3′-dideoxy-2′,3′-didehydrocytidine derivatives for use in treating HIV and hepatitis B (“HBV”).
Due to the importance of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides, and in particular, β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (“β-D-D4FC”), as potent antihuman immunodeficiency virus agents, there is a need for a commercial or industrial scale process for the synthesis of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides, and in particular, β-D-D4FC. On addition, natural and unnatural D4 nucleosides can serve as synthetic intermediates for the preparation of a large variety of other nucleoside analogs, including but not limited to 2′,3′-dideoxy, and 2′ or 3′-deoxyribo-nucleoside analogs as well as additional derivatives obtained by subsequent functional group manipulations. In view of the importance of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides, it is desirable to have a facile, efficient and general route of synthesis of these compounds. While several methods exist for the synthesis of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides, none has the ability to produce efficiently both enantiomeric forms of these compounds using purine bases, pyrimidine bases, heteroaromatics or heterocycles.
Several syntheses for the preparation of β-D-D4FC or its enantiomer, β-L-D4FC, have been reported. See Schinazi R. F. et al, J. Med. Chem. 1999, 42,859-867; Chen S., Biorganic & Medicinal Chemistry Letters 8(1998) 3245-3250; Doyle, T. W., et al., J. Org. Chem. 1997,62, 3449-3452; Cheng, Y., et al., J. Med. Chem. 1996, 62, 1757-1759; and Lin et al., U.S. Pat. No. 5,561,120.
One of the earliest syntheses of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides is the published process for the preparation of 2′,3′-dideoxy-2′,3′-didehydro-thymidine (D4T). The first reported method to produce D4T involved the base promoted elimination of 3′-O-sulfonyl esters of 2′-deoxynucleosides. This synthetic route is limited to pyrimidine nucleosides and cannot be used in the production of purine nucleosides. Horwitz, J. P., et al., J. Org. Chem. 1966, 31, 205; Horwitz, J. P., et al., J. Org. Chem. 1967, 32, 817; and Horwitz, J. P., et al., J. Am. Chem. Soc. 1964, 86, 1896.
Some 2′,3′-dideoxy-2′,3′-didehydro-nucleosides have been obtained directly from the corresponding ribonucleosides through their reaction with acetoxyisobutyryl 5 halides, followed by the reductive elimination of the 2′,3′-acetoxy-2′,3′-halogeno derivatives with chromous ion. U.S. Pat. No. 3,817,982 (1974); Chem. Abstr. 1974, 81, 63942; Russell, A. F., et al., J. Am. Chem. Soc. 1973, 95, 4025; Jain, T. C., et al., J. Org. Chem. 1974, 39, 30; Classon, B., et al., Acta Chem. Scand. Sect B 1982, 32, 251; Robins, M. J., et al., Tetrahedron Letters 1984, 25, 367. In a variation of this method, zinc in dimethylformamide can be used instead of chromous acetate. Robins, M. J., et al., Tetrahedron Letters 1984, 25, 367. The reaction is difficult, and results in several products, and is therefore an inefficient route to obtain the 2′,3′-unsaturated compounds (Jain, T. C., et al., J. Org. Chem. 1974, 39, 30).
U.S. Pat. No. 5,455,339 to Chu describes a method for preparing 2′,3′-dideoxy-2′,3′-didehydro-nucleosides via dialkyl xanthate intermediates including:                (i) preparing a nucleoside derivative of the general structure:          wherein B is a nitrogen, oxygen, or sulfur heterocycle of from C1 to C15, Y is a suitable oxygen protecting group, each R is C(S)SR′, where R1 is an alkyl or cyanoalkyl group of C1 to C15, or both R's together are >C═S;        (ii) activating the 2′ and 3′ hydroxyls to form 2′,3′-thiocarbonates; and then        (iii) deoxygenating the nucleoside derivative to the corresponding 2′,3′-dieoxy-2′,3′-didehydronucleoside.        
U.S. Pat. Nos. 5,703,058; 5,905,070 and 6,232,300 and International Patent Application No. PCT/US96/00965, published as WO 96/22778 to Raymond F. Schinazi and Dennis C. Liotta describes [5-carboxamido or 5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleosides and [5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleosides. Example 3 of the '070 patent provides a process for the preparation of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides. The patent states that the procedure can be adapted for a wide variety of bases and can be used to provide either the β-D or the β-L isomer as desired. The process is illustrated below: wherein MMPP is magnesium monoperoxyphthalate, R is t-butyldiphenylsilyl, and STIPP is 2,4,6-triisopropylphenyl.
PCT WO 99/43691 describes 2′-fluoro-2′,3′-dideoxy-2′,3′-didehydronucleosides that are useful in the treatment of viral infections. Schemes 9, 10, and 11 of the PCT describe methods for the preparation of β-L-2′-fluoro-2′,3′-didehydro-2′,3′-dideoxy-nucleosides. The PCT publication states that previously, the synthesis of 2′,3′-unsaturated L-nucleosides had been accomplished via an elimination reaction starting from readily available nucleoside analogs, which involved a lengthy modification procedure. There are few examples of the synthesis of 2′,3′-unsaturated purine nucleosides by direct condensation due to the lability of the 2′,3′-unsaturated sugar moiety under the coupling conditions in the presence of a Lewis acid, except one case of the pyrimidine analog using a thiophenyl intermediate (Abdel-Medied, A. W.-S., et al., Synthesis, 1991, 313; Sujino, K., et al., Tetrahedron Lett., 1996, 37, 6133). In contrast to the 2′,3′-unsaturated sugar moiety, the 2′-fluoro-2′,3′-unsaturated sugar, which bears enhanced stability of the glycosyl bond during the condensation with a heterocycle, is more suitable for the direct coupling reaction. As illustrated below (wherein B is a purine or pyrimidine base and R is an oxygen protecting group), (R)-2′-fluorobutenolide (prepared from L-glyceraldehyde acetonide) was used as the key precursor in the preparation of 2′-fluoro-2′,3′-dideoxy-2′,3′-didehydro-nucleosides. From the acetonide, a mixture of E and Z isomers was obtained via the Homer-Emmons reaction in the presence of triethyl α-fluorophosphonoacetate and sodium bis(trimethylsilyl)amide in THF (Thenappan, A., et al., J. Org. Chem., 1990, 55, 4639; Morikawa, T., et al., Chem. Pharm. Bull., 1992, 40, 3189; Patrich, T. B., et al., J. Org. Chem., 1994, 59, 1210). Due to the difficulties in separating the E and Z isomers, the mixture was carried on to the cyclization reaction under acidic conditions to give the desired lactone and uncyclized diol. The resulting mixture was converted to the silyl lactone and was subjected to reduction with DIBA1-H in CH2Cl2 at 78° C. to give the lactol. The lactol was treated with acetic anhydride to yield a key acetate intermediate, which was condensed with silylated 6-chloropurine under Vorbruggen conditions to afford anomeric mixtures of the protected nucleoside. Treatment of the protected nucleoside with TBAF in THF gave a mixture of free nucleosides that could be separated by silica gel column chromatography. The adenine analogs are obtained by the treatment of 6-chloropuridine with mercapto-ethanol and NaOMe in a steel bomb at 90° C. Further treatment of the adenine analogs under the same conditions afforded the inosine analogs. 
The above methods for the syntheses of β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, or its enantiomer, β-L-D4FC, disclose synthetic routes employing combinations of toxic and/or difficult to handle reagents, linear or sequential reaction steps, and laborious chromatographic or purification steps. Consequently, these syntheses afford an inefficient synthesis of β-D-D4FC.
Therefore, it is another object of the present invention to provide a process for the production of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides that is facile and efficient.
It is an object of the present invention to provide a high yield method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular p-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine.
It is an object of the present invention to provide a method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, which produces few undesired side products.
It is another object of the present invention to provide a method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, which is amenable to commercial production.
It is a particular object of the present invention to provide a method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, which does not require protection of the purine or pyrimidine base, such as cytosine or 5-fluorocytosine.
It is a particular object of the present invention to provide a method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, which does not require purification by chromatography.
It is a particular object of the present invention to provide a method to manufacture 2′,3′-didehydro-2′,3′-dideoxynucleosides, and in particular p-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, which does not require the use of toxic metals.
It is a further object of the present invention to provide a process for the production of 2′,3′-dideoxy-2′,3′-didehydro-nucleosides that can be used as synthetic intermediates for the preparation of a large variety of other nucleoside analogs, including but not limited to 2′,3′-dideoxy, and 2′ and 3′-deoxyribo nucleoside analogs as well as additional derivatives obtained by subsequent functional group manipulations.